Several methods are known for direct determination of iron in blood serum. Some of these methods are based on the possibility to form colored complexes between divalent iron ion and complexing agents such as BP (4,7-diphenyl-1,10-phenantroline), TPTZ (2,4,6-tris(2-pyridyl)-s-triazine) and ferrozine [disodium salt of 3-(2-pyridyl)-5,6-bis (4-sulfophenyl)-s-triazine; See L. L. Stookey: Analytical Chemistry, 42, No. 7, 779, 1970] and to determine spectrophotometrically the complex formed. Specific literature states that ferrozine is particularly suitable in that it forms with divalent iron a colored complex of very high absorbancy (.epsilon.=27.100) soluble in water and stable at pH between 3.5 and 11 and preferably between 4 and 9.
Most of the methods for determination of iron is serum described in the prior art require (1) dissociation of iron from serum transferrin by treatment with strong mineral acids in the presence of a reducing agent (2) deproteinization of serum through precipitation of proteins, for example, with trichloacetic acid, (3) determination of the bivalent iron ion remaining in the solution through color reaction with ferrozine after the pH of the solution is brought at a value between 3 and 6 with buffer (See P. Carter: Anal. Biochem. 40, 450, 1971).
Other methods are known as "direct" methods, which do not require precipitation of the proteins (See for instance J. P. Persijn et al., Clin. Chim. Acta 35, 91, 1971; J. M. White et al., Clin. Chem. Vol. 19, No. 5, 526, 1973; R. Ruutu: Clin. Chim. Acta 61, 229, 1975).
These methods, generally involve use of buffers in a pH range corresponding to the isoelectric point of most of serum proteins and therefore to their maximum of instability. Precipitation of proteins that would strongly interfere with analytical results is obviated by addition of detergents. These should also operate the detachment of iron from transferrin without lowering the pH. However the variable proteins composition of the various sera does not allow an uniform and stable protection against turbidity formation in every case. In fact, individual differences are not obviated by the reading of the initial absorbance or of the blank in that, in several instances, an unpredictable turbidity may be superimposed to the colored complex. Furthermore the release of iron from transferrin may not be complete giving erroneously low values. For the above reasons the Expert Panel on Iron of the International Committee for Standardization in Haematoglogy has rejected as reference serum iron method all of the numerous "direct" methods without proteins precipitation so far known. (See E. W. Rice et al., Clin. Chim. Acta 53, 391, 1974).
On the other hand, serum deproteinization sensibly lengthens the procedure and constitutes a critical step since serum filtrates, after proteins precipitation, may not be perfectly clear; moreover, a concentrated buffer is required to adjust the pH in a range suitable for the chromogenic reaction and this may be a source of iron contamination. The presence of copper that, under certain pathological circumstance may reach very high levels, can interfere strongly on spectrophotometrical methods, especially those based on the use of ferrozine. (See Hugh et al., Clin. Chem. Vol. 17, No. 9, 950, 1971; J. R. Duffy et al., Clin. Biochem. 10, 122, 1977).